Apoptosis, or programmed cell death, can be triggered by extrinsic or intrinsic signals, as well as developmental cues. Two major apoptotic pathways have been identified: an extrinsic cell death pathway in which apoptosis is initiated through ligand binding to cell surface receptors expressed on the cell that will subsequently die, and an intrinsic cell death pathway in which apoptosis is initiated within the cell.
Tumor necrosis factor receptor family members are among the best characterized of receptors involved in the extrinsic death pathway. Upon the engagement of ligand, these receptors initiate the formation of a death-inducing signaling complex, which includes an essential adaptor molecule FADD. FADD in turn recruits Caspase 8, which in turn is activated through an autoproteolytic process. Once activated, Caspase 8 initiates both the activation of additional caspases such as Caspase 9 and Caspase 3, and the degradation of intracellular substrates, which eventually results in cell death. The TNF receptor family includes TNFR1; Fas; DR3 proteins such as Apo3, WSL-1, TRAMP, and LARD; DR4; DR5 proteins such as TRAIL-R2, TRICK2, and KILLER; and DR6.
Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues, such as Bcl-xl, inhibit apoptosis, whereas other family members, such as Bax and Bak, induce or accelerate apoptosis under certain conditions. Bak and Bax, as well as Bcl-xs, Bid, Bim, Bad and Bik, constitute the pro-apoptotic group of Bcl-2 proteins.
The Bax protein shares highly conserved domains with Bcl-2, some of which are required for the formation of Bax/Bcl-2 heterodimers, which are thought to be important for the survival or death response to apoptotic signals. Following its activation, Bax translocates to the outer mitochondrial membrane where it oligomerizes, renders the membrane permeable, and releases several death-promoting factors, including cytochrome C (Scorrano et al. (2003) Biochem. Biophys. Res. Commun. 304:437-444). Bax can be rendered inactive in normal cells via interaction with the Ku70 protein, which sequesters Bax from mitochondria (Sawada et al. (2003) Nat. Cell Biol. 5:320-329).
Bak also promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Like Bax, the Bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Bak is a potent inducer of apoptosis in various cell types.
Cell lines in which expression of Bax and/or Bak is reduced by introduction of antisense molecules have been described. See, e.g., Mandic et al. (2001) Mol. Cell Biol. 21:3684-3691. In addition, Bax and/or Bak deficient fibroblasts have also been immortalized to create cell lines. See, U.S. Patent Application No. 20030091982. Furthermore, naturally occurring zinc finger proteins (Grimes et al. (1996) Proc. Nat'l. Acad. Sci. USA 93:14569-14573) fused to repression domains and engineered zinc finger proteins and nucleases (see, U.S. Patent Publication No. 20060063231) have also been described for the inactivation of Bax and/or Bak. Engineered zinc finger nucleases have also been used to inactivate endogenous dihydrofolate reductase (dhfr) and CCR5 genes. See, e.g., U.S. Patent Publication No. 20080015164 and PCT Publication WO 2007/139982.
However, there remains a need for methods and compositions for inactivation of Bax and Bak, for example to facilitate generation of apoptosis-resistant cell lines.